Development of controlled release systems of biological factors for the regeneration of intervertebral disc

Abstract

Purpose: The degeneration of intervertebral disc (IVD), and notably of its central part the nucleus pulposus (NP), is responsible for 40% of chronic low back pain (LBP). Recent studies have reported the existence of endogenous regenerative cells in the IVD vicinity and within the IVD. These endogenous cells have been characterized as mesenchymal stem cell-like progenitors residing in specific niches. In response to chemokines (stromal derived factor-1 (SDF-1)) these cells can be recruited and migrate to the site of injury and thus contribute to the endogenous repair process. In this context, the development of microbeads-based local delivery systems of factors involved in progenitors recruitment has recently been contemplated for IVD regenerative medicine. Because of their physicochemical and biological properties, microbeads of pullulans (PMBs) have long been investigated as drug carriers. In addition, we recently demonstrated that transforming growth factor-β1 (TGF-β1) and growth differentiation factor5 (GDF5) are potent stimulators of the differentiation of mesenchymal stem cells (MSC) into NP-like cells. In this context, the aim of this work was to develop an intradiscal pullulan microbeads-based delivery system for the controlled release of SDF-1, TGF-β1 and GDF5. This drug delivery system would be able to sequentially contribute to 1) the recruitment and mobilization of resident progenitors, 2) the differentiation of the mobilized progenitors and 3) the subsequent regeneration of NP. Methods: Chemotaxis assays were performed to determine the in vitro cell migration. Human MSCs (1250 cells/μl) were incubated with or without SDF-1 (250 ng/ml) in Transwells for 4h, migratory cells were stained by crystal violet then quantified by spectrophotometry. In parallel, PMBs were prepared by a simultaneous crosslinking protocol coupled to a water-in-oil emulsification process. Freeze-dried PMBs were incubated with GDF-5 and TGF-β1 separately (25 mg of PMBs at final concentration of 1, 2 and 4 μg/mL and in a final volume of 500 μL of PBS) for 24 h at 4°C under rotary stirring at 24 rpm. GDF-5 and TGF-β1 release assays were performed in PBS at 37°C for 21 days and concentrations were measured by ELISA. Results: SDF-1 has improved the in vitro migration of hMSCs, increasing by more than twice the number of migratory cells. GDF-5 and TGF-β1 were successfully adsorbed on PMBs with a 100% efficiency. Release experiments showed a burst release within the 1st h, at 604 ng/h and 50 ng/h for GDF-5 and TGF-β1 respectively, then the release rate decreased during 21 days with 0.6 ng/h and 0.15 ng/h during the last 7 days for GDF-5 and TGF-β1, respectively. At day 21, GDF-5 was entirely released, whereas only 40% of TGF-β1 was released. This different release profiles could be explained by the difference of molecular weight (13 kDa for GDF-5 and 25 kDa for TGF-β1). Conclusions: We have confirmed that SDF-1 improved hMSCs in vitro migration, and that PMBs are suitable microcarriers for the loading and release of GDF-5 and TGFβ-1. The loading and release capability of SDF-1 by PMBs, as well as SDF-1 bioactivity after release will be analyzed, to obtain a fast and massive recruitment of resident progenitors in vivo. Then, we will study the action of GDF-5 and TGFβ1 released from PMBs on in vitro NP differentiation, by using a 3D matrix model to mimic the NP microenvironment. Nucleopulpogenic differentiation will be evaluated by analysis of specific extracellular matrix production and gene expression markers.